Urine-derived mesenchymal stem cells-derived exosomes enhances survival and proliferation of aging retinal ganglion cells

Effects of D-galactose on survival rate of RGC cells

The cell viability of RGCs treated with different concentrations of D-galactose was detected by CCK8 assay. As demonstrated in Table 1, the cell survival rate was negatively correlated with the concentrations of D-galactose. With the concentration of D-galactose increasing, the survival rate gradually decreased, reaching about 58% when treated with 30 mg/mL D-galactose. The survival rate at 30 mg/mL was significantly lower than that of control group (p < 0.001, Fig. 1). Thus, 30 mg/mL D-galactose was determined to be the final concentration of inducing aging RGCs.

Table 1 Survival rate of RGCs treated with different concentrations of D-galactoseFig. 1figure 1

Survival rate of RGCs treated with different concentrations of D-galactose. N = 8 /group, ***p < 0.001

Identification of USCs

Immunostaining results demonstrated that over 99% positive expression of CD44 and CD90 identified the purity of cultured USCs (Fig. 2). The high percentage of cells expressing CD44 and CD90 indicates MSCs characteristics of USCs.

Fig. 2figure 2

Immunostaining of CD44 and CD90 for USCs identification. Green fluorescence indicates CD44 positive cells, and red fluorescence indicates CD90 positive cells. Scale bar = 100 μm, n = 6 /group

Apoptosis of aging RGCs was inhibited after USCs conditioned medium treatment

Flow cytometry was employed to detect the apoptosis rate of RGCs. The results revealed that apoptotic RGCs were significantly increased in the aging group, while the number of apoptotic cells got smaller in the aging RGCs cultured in USCs medium (Fig. 3A, B). Correspondingly, the apoptosis rate in aging group was much higher than that of normal group (p < 0.01, Fig. 3C), but it was markedly diminished in RGCs treated with USCs medium (p < 0.01, Fig. 3C). The results revealed that USCs-derived exosomes could inhibit the apoptosis of aging RGCs. Further, the cell count of RGCs in aging group at Freq G1 was largely increased, but obviously decreased at Freq S compared with that of the normal group (Fig. 3D, E). No obvious variation was revealed in the number of cells at Freq G2 among normal, aging and USCs medium groups (Fig. 3D, E). Relative to normal group, there’s a prominent increase of the cell number observed at Freq G1 in aging group, while a significant reduction at Freq G1 in USCs medium group (Fig. 3D, E).

Fig. 3figure 3

Apoptosis and cell cycle of RGCs among normal, aging and USCs medium groups. A Light field images of RGCs in normal group, aging group and aging + USCs medium group, scale bar = 100 μm. B The flow cytometry detection and C the apoptosis rate of RGCs among normal, aging and aging + USCs medium groups. D The cell cycle and E cycle rate of RGCs among normal, aging and aging + USCs medium groups. All data are shown as the mean ± SD, n = 6 /group. **p < 0.01

USCs conditioned medium promoted survival and proliferation of aging RGCs

Three kinds of medium (DMEM; USCs conditioned medium; DMEM to USCs conditioned medium at 1:1) were used to culture RGCs in normal and aging groups. Normal RGCs in DMEM are irregular hexagon-shaped with uniform size, growing in good state (Fig. 4A). When cultured in USCs conditioned medium, the number of normal RGCs was increased and arranged more closely. In the combined medium with DMEM and USCs conditioned medium, variation in cell morphology was observed and cell number was reduced (Fig. 4A). When aging RGCs were cultured in DMEM, the cell count was significantly decreased relative to that of normal RGCs in DMEM (p = 0.00, Fig. 4B). However, aging cells in USCs conditioned medium were augmented markedly compared to cells in DMEM medium (p = 0.00, Fig. 4A, B) and combined medium. Cell viability was detected using CCK8 test at 24 h and 48 h, which demonstrated that in DMEM medium, the OD value of aging RGCs significantly reduced compared to that of normal RGCs at 24 h and 48 h (p = 0.00), while aging RGCs in USCs conditioned medium showed obvious augmented OD value in comparison to aging RGCs in DMEM at 24 h and 48 h (p = 0.00, Fig. 4C, D). The results of β-Galactosidase staining uncovered that there were more aging cells in DMEM medium, decreased number of aging RGCs and increased number of normal cells when cultured in USCs conditioned medium (Fig. 4E).

Fig. 4figure 4

The effect of USCs conditioned medium on ameliorating aging RGCs. A Light field images of RGCs in normal and aging groups cultured in three kinds of culture medium, respectively, scale bar = 100 μm. B The cell number of RGCs in three kinds of culture medium between normal and aging groups, n = 12/group. OD value of RGCs in normal + DMEM medium, normal + USCs medium, aging + DMEM medium and aging + USCs medium groups at (C) 24 h and (D) 48 h, n = 6/group. E β-Galactosidase staining images of RGCs in normal + DMEM medium, aging + DMEM medium and aging + USCs medium groups, scale bar = 100 μm, n = 6/group

Identification of DEGs and analysis on their biological functions

In order to further investigate the underlying mechanisms related to the effect of USCs-derived exosomes on aging RGCs, the gene sequencing was performed to analyze the potential genetic variation. The sequencing outcomes demonstrated 117 upregulated genes and 186 downregulated genes in normal RGCs group vs aging RGCs group, 137 upregulated ones and 517 downregulated ones in aging RGCs group vs aging RGCs + USCs medium group (Fig. 5A). These DEGs underwent subsequent GO and KEGG analysis. As revealed, the top 20 enriched KEGG pathways involved by DEGs in aging RGCs vs normal RGCs were presented: Cholesterol metabolism Biosynthesis of amino acids, Complement and coagulation cascades, ABC transporters Metabolic pathways, Seleno compound metabolism, Steroid biosynthesis Osteoclast differentiation, Prion diseases, Alanine, aspartate and glutamate metabolism, Ovarian Steroidogenesis p53 signaling pathway, Glycine, Serine and threonine metabolism, Chemical carcinogenesis, Synaptic vesicle cycle, Pertussis, One carbon pool by folate, Ether lipid metabolism Arginine biosynthesis, Dorso-ventral axis formation (Fig. 5B). Meanwhile, the top 20 enriched KEGG pathways related to DEGs in aging RGCs vs aging cells cultured in USCs conditioned medium were Cytokine-cytokine receptor interaction (map04060), Jak-STAT signaling pathway (map04630), Circadian entrainment (map04713), Proximal tubule bicarbonate reclamation (map04964), MicroRNAs in cancer (map05206), Viral protein interaction with cytokine and cytokine receptor (map04061), Chemokine signaling pathway (map04062), Mineral absorption (map04978), Protein digestion and absorption (map04974), Apelin signaling pathway (map04371), TNF signaling pathway (map04668), Aldosterone-regulated sodium reabsorption (map04960), Estrogen signaling pathway (map04915), Glycine, serine and threonine metabolism (map00260), Axon guidance (map04360), PPAR signaling pathway (map03320), Other types of O-glycan biosynthesis (map00514), Insulin resistance (map04931), IL-17 signaling pathway (map04657), Relaxin signaling pathway (map04926). In addition, the GO terms DEGs involved are also displayed (Fig. 5C). Top 10 biological processes in which DEGs in aging RGCs vs normal cells participated included cellular process, biological regulation, regulation of biological process, metabolic process, response to stimulus, multicellular organismal process, negative regulation of biological process, developmental process, localization, positive regulation of biological process (Fig. 5D). Cell components of these DEGs include cell, cell part, organelle, membrane, organelle part, membrane part, extracellular region. Molecular functions of these DEGs involved mainly include binding, catalytic activity, molecular function regulator, transporter activity (Fig. 5D). Comparatively, top 10 biological processes in which DEGs in aging cells vs aging RGCs + USCs medium participated involved cellular process, biological regulation, metabolic process, regulation of biological process, response to stimulus, multicellular organismal process, developmental process, negative regulation of biological process, signaling, positive regulation of biological process (Fig. 5E). Cell components of these DEGs included cell, cell part, organelle, membrane, membrane part, organelle part, extracellular region, membrane-enclosed lumen. Molecular functions of these DEGs involved binding, catalytic activity, molecular function regulator, transporter activity (Fig. 5E).

Fig. 5figure 5

Bioinformatics analysis of DEGs involved in the effect of USCs-derived exosomes on aging RGCs. A The number of DEGs and volcano maps in normal RGCs group vs aging RGCs group, and aging RGCs group vs aging RGCs + USCs medium group, n = 3/group. B, C The enriched KEGG pathways involved by DEGs. D, E GO terms enriched by DEGs

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